Characterization of the Glycosylation Sites in Cyclooxygenase-2 Using Mass Spectrometry

Nemeth, Jennifer F.; Hochensang, G. Phillip; Marnett, and Lawrence J.; Caprioli, Richard M.

doi:10.1021/bi002313c

Cited by 75 publications

(47 citation statements)

References 25 publications

(43 reference statements)

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“…is an N-glycoprotein with 4 asparagine (Asn) sites, and if N-glycosylated, COX-2 is expressed in doublets, 72 and 74 kDa, with full enzymatic activities (15,16). The mass difference between 72 and 74 kDa COX-2 is suggested to be due to variance of the degree of N-glycosylation on Asn 580 (15).…”

Section: Gs-hcl-mediated Decrease In Cox-2 Molecular Mass Is Associatmentioning

confidence: 99%

“…The mass difference between 72 and 74 kDa COX-2 is suggested to be due to variance of the degree of N-glycosylation on Asn 580 (15). Interestingly, it has been recently shown that GS prevents N-glycosylation of certain proteins (29,30), including glucose transporter 1 and a lipoprotein apoB-100.…”

Section: Gs-hcl-mediated Decrease In Cox-2 Molecular Mass Is Associatmentioning

confidence: 99%

“…In addition, activities of MAPKs, including ERKs, p38 MAPK, and JNKs, were reported to be important for COX-2 expression (13,14). COX-2 is an N-glycoprotein with four glycosylation sites (15,16). Of interest, it has been previously shown that inhibition of COX-2 N-glycosy-lation by site-directed mutagenesis or tunicamycin (TN), a protein N-glycosylation inhibitor, results in expression of COX-2 with the reduced molecular mass and activity (17), indicating the importance of this co-translational modification in COX-2 enzyme catalysis.…”

mentioning

confidence: 99%

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Glucosamine Hydrochloride Specifically Inhibits COX-2 by Preventing COX-2 N-Glycosylation and by Increasing COX-2 Protein Turnover in a Proteasome-dependent Manner

Jang

Sung

Park

et al. 2007

Journal of Biological Chemistry

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COX-2 and its products, including prostaglandin E 2 , are involved in many inflammatory processes. Glucosamine (GS) is an amino monosaccharide and has been widely used for alternative regimen of (osteo)arthritis. However, the mechanism of action of GS on COX-2 expression remains unclear. Here we describe a new action mechanism of glucosamine hydrochloride (GS-HCl) to tackle endogenous and agonistdriven COX-2 at protein level. GS-HCl (but not GS sulfate, N-acetyl GS, or galactosamine HCl) resulted in a shift in the molecular mass of COX-2 from 72-74 to 66 -70 kDa and concomitant inhibition of prostaglandin E 2 production in a concentration-dependent manner in interleukin (IL)-1␤-treated A549 human lung epithelial cells. Remarkably, GS-HCl-mediated decrease in COX-2 molecular mass was associated with inhibition of COX-2 N-glycosylation during translation, as assessed by the effect of tunicamycin, the protein N-glycosylation inhibitor, or of cycloheximide, the translation inhibitor, on COX-2 modification. Specifically, the effect of low concentration of GS-HCl (1 mM) or of tunicamycin (0.1 g/ml) to produce the aglycosylated COX-2 was rescued by the proteasomal inhibitor MG132 but not by the lysosomal or caspase inhibitors. However, the proteasomal inhibitors did not show an effect at 5 mM GS-HCl, which produced the aglycosylated or completely deglycosylated form of COX-2. Notably, GS-HCl (5 mM) also facilitated degradation of the higher molecular species of COX-2 in IL-1␤-treated A549 cells that was retarded by MG132. GS-HCl (5 mM) was also able to decrease the molecular mass of endogenous and IL-1␤-or tumor necrosis factor-␣-driven COX-2 in different human cell lines, including Hep2 (bronchial) and H292 (laryngeal). However, GS-HCl did not affect COX-1 protein expression. These results demonstrate for the first time that GSHCl inhibits COX-2 activity by preventing COX-2 co-translational N-glycosylation and by facilitating COX-2 protein turnover during translation in a proteasome-dependent manner.

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Section: Gs-hcl-mediated Decrease In Cox-2 Molecular Mass Is Associatmentioning

confidence: 99%

Section: Gs-hcl-mediated Decrease In Cox-2 Molecular Mass Is Associatmentioning

confidence: 99%

mentioning

confidence: 99%

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Glucosamine Hydrochloride Specifically Inhibits COX-2 by Preventing COX-2 N-Glycosylation and by Increasing COX-2 Protein Turnover in a Proteasome-dependent Manner

Jang

Sung

Park

et al. 2007

Journal of Biological Chemistry

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“…COX-2 was visualized on Western blot either as a single-band or a multiple-band product (Figure 1). Multiple bands likely corresponded to COX-2 glycosylation products (Nemeth et al, 2001). In order to identify the molecular mechanisms responsible for COX-2 expression induced by a sublethal PDT dose, we used 5 mM PPME in combination with a 3.2 J/ cm 2 fluence, corresponding to a 95% survival rate after 24 h (data not shown).…”

Section: Ppme-pdt Induces Cox-2 Expressionmentioning

confidence: 99%

Distinct transduction mechanisms of cyclooxygenase 2 gene activation in tumour cells after photodynamic therapy

et al. 2005

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“…The presence of doublet bands is reported to be due to differential glycosylation on COX-2 protein. 34) We selected liver, small intestine and colon for the assessment of peripehral COX-2 induction upon LPS or zymosan injection. In liver from mice treated with vehicle, COX-2 expression was not detectable but was elevated upon zymosan injection.…”

Section: Anorexic Response After Zymosan or Lps Injectionmentioning

confidence: 99%

Differential Effects of Selective Cyclooxygenase (COX)-1 and COX-2 Inhibitors on Anorexic Response and Prostaglandin Generation in Various Tissues Induced by Zymosan

Naoi

Kogure

Saito

et al. 2006

Biological & Pharmaceutical Bulletin

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We have shown that anorexic response is induced by intraperitoneal injection of zymosan in mice, although the role of prostaglandins in this response is relatively unknown as compared with lipopolysaccharide (LPS)-induced anorexic response. Indomethacin (0.5 and 2.0 mg/kg), a non-selective cyclooxygenase (COX) inhibitor, as well as meloxicam (0.5 mg/kg), a selective COX-2 inhibitor, but not FR122047 (2.0 mg/kg), a selective COX-1 inhibitor, attenuated zymosan-induced anorexia. Zymosan injection elevated COX-2 expression in brain and liver but not in small intestine and colon. Meloxicam (0.5 mg/kg) and FR122047 treatment (2.0 mg/kg) similarly suppressed the generation of brain prostaglandin E 2 (PGE 2 ) and peritoneal prostacyclin (PGI 2 ) upon zymosan injection. PGE 2 generation in liver upon zymosan injection was suppressed by meloxicam (0.5 mg/kg) but not by FR122047 treatment (2.0 mg/kg). Our observations suggest that COX-2 plays an important role in zymosan-induced anorexia, which is a similar feature in LPS-induced anorexic response. However, non-selective inhibition by selective COX-1 and COX-2 inhibitors of brain PGE 2 generation upon zymosan injection does not support the role of COX-2 expressed in brain in zymosan-induced anorexic response. PGE 2 generation in liver may account for peripheral role of COX-2 in zymosan-induced anorexic response.

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Characterization of the Glycosylation Sites in Cyclooxygenase-2 Using Mass Spectrometry

Cited by 75 publications

References 25 publications

Glucosamine Hydrochloride Specifically Inhibits COX-2 by Preventing COX-2 N-Glycosylation and by Increasing COX-2 Protein Turnover in a Proteasome-dependent Manner

Glucosamine Hydrochloride Specifically Inhibits COX-2 by Preventing COX-2 N-Glycosylation and by Increasing COX-2 Protein Turnover in a Proteasome-dependent Manner

Distinct transduction mechanisms of cyclooxygenase 2 gene activation in tumour cells after photodynamic therapy

Differential Effects of Selective Cyclooxygenase (COX)-1 and COX-2 Inhibitors on Anorexic Response and Prostaglandin Generation in Various Tissues Induced by Zymosan

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